Design and synthesis of new fluconazole analogues

Article abstract of DOI:10.1039/c5ob00590f

We have synthesized new fluconazole analogues containing two different 1,2,3-triazole units in the side chain. The synthesis of new amide analogues using a variety of acids is also described. All the compounds showed very good antifungal activity. A hemol

Full text of DOI:10.1039/c5ob00590f

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DOI: 10.1039/C5OB00590F
Journal Name
ARTICLE
Design and Synthesis of new fluconazole analogues
Vandana S. Pore,a*Sandip G. Agalave,a Pratiksha Singh,b Praveen K. Shukla,b Vikash Kumar,c
Mohammad I. Siddiqic
Received 00th January 20xx,
Accepted 00th January 20xx
Abstract: We have synthesized new fluconazole analogues containing two different 1,2,3-triazole units in the side chain.
Synthesis of new amide analogues by using a variety of acids is also described. All the compounds showed very good
antifungal activity. Hemolysis study of the most active compounds 6e and 13j showed that both compounds did not cause
any hemolysis at the dilutions tested. These compounds did not exhibit any toxicity to L929 cells at MIC and lower
concentrations. In docking study, overall binding mode of 6e and 13j appeared reasonable and provided a good insight
into the structural basis of inhibition of Candida albicans Cyp51 by these compounds.
DOI: 10.1039/x0xx00000x
www.rsc.org/
Introduction
Invasive fungal disease continues to be a problem associated with
significant morbidity and high mortality in immune compromised
and, to a lesser extent, immune competent individuals1,2. In many
cases, it is not the AIDS or cancer itself but the mycoses that are
lethal to these patients. Serious fungal infections are caused mostly
by Candida albicans, Cryptococcus neoformans and Aspergillus
fumigates3,4. The common antifungal agents currently used in clinic
are polyenes, allylamines, azoles and echinocandins5. The current
antifungal drugs like amphotericin B are highly toxic. Some drugs
such as flucytosine and azoles are becoming ineffective due to
appearance of resistant strains.
Triazole antifungals have emerged as front-line drugs for the
treatment and prophylaxis of many systemic mycoses because of
their broad antifungal spectrum, high potency and low toxicity
Figure 1- Presently marketed Azole antifungals containing 1,2,4-triazole
triazoles such as voriconazole, posaconazole, ravuconazole,
albaconazole etc. containing 1,2,4-triazole and difluorobenzene
moieties are marketed or in the late stages of clinical trials (Figure
1)10.
against most yeasts and fungi.6
A large number of triazole
compounds as clinical drugs or candidates have been frequently
employed for the treatment of various types of diseases, which
have shown their large development value and wide potential as
medicinal agents. There are several reports on current
developments of azole-based antifungal drugs.7 Unfortunately, the
excessive use of azoles has led to development of severe resistance,
which significantly reduced the efficacy of them.8,9 Several new
Fluconazole, α-(2,4-difluorophenyl)-α-(1H-1,2,4 triazol-1-yl-methyl)-
1H-1,2,4 triazol-1-ethanol (Figure 1) plays an excellent role in
prophylaxis, empirical therapy, and the treatment of both
superficial and invasive yeast fungal infections. It is a potent
inhibitor of the cytochrome P450 (CYP)-mediated metabolism of the
antiepileptic agent phenytion, a well-known human and animal
tetratogen11. Fluconazole is well absorbed and exhibit high oral
bioavailability. It is an antifungal agent of choice for the treatment
of infections by Candida albicans and Cryptococcus neoformans due
to its potent activity, excellent safety profile, and favourable
pharmacokinetic characteristics12. However fluconazole is not
fungicidal and is ineffective against invasive aspergillosis. Extensive
use of fluconazole has increased the number of fluconazole-
resistant C. albicans isolates13. Therefore, toxicity concerns, limited
aOrganic Chemistry Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha
Road, Pashan, Pune 411 008, India.
bMedical Mycology Lab., Division of Fermentation Technology, CSIR-Central Drug
Research Institute, Lucknow, 226001, India
cComputational Bio & Bioinformatics Lab., Division of Molecular &Structural Biology,
CSIR-Central Drug Research Institute, Lucknow, 226001, India
Electronic Supplementary Information (ESI) available: 1H and 13C NMR of all the
compounds. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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N
OH
antifungal spectrum, development of resistant strains and observed
nephrotoxicity to fluconazole have created a need to modify its
activity. Several reports on the synthesis and biological activity of
structurally modified new analogues of fluconazole are available in
the literature14.
N
O
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DOI: 10.1039/C5OB00590F
N
F
N
F
N
N
a
2
1
F
F
R
c, d
b
e
NaN3
HCHO
N
R
N
N
3 (a-e)
The cytochrome P450 (CYP) enzymes play an essential role in the
metabolism of xenobiotics by catalyzing the monooxygenation of a
broad diversity of substrates. Some nitrogen-containing
heteroaromatic xenobiotics such as pyridine, imidazole, and triazole
derivatives are known to inhibit CYP enzymes by direct coordination
with the heme iron (type II ligands).15 Based on the structure of the
active site of CYP51 and the extensive investigation of the structure
activity relationships of azole antifungals, we designed and
R
OH
2,4-disubstituted
1,2,3-triazole
4 (a-e)
N
N
OH
N
R
N
OH
N
F
N
N
F
N
N
N
N
N
a, R = Ph
b, R = C6H13
c, R = p-fluorophenyl
d, R = p-t-butylphenyl
e, R = cyclohexylmethyl
N
N
N
N3
F
2
5 (a-e)
6 (a-e)
F
Reagents and conditions: (a) Zn, propargyl bromide, DMF/THF, 25
h, 95%; (b) i) AcOH, 1,4-dioxane; ii) CuSO4·5H2O (5 mol%), Sodium
ascorbate (40 mol%), 16-18h, (R= Ph 92%, R= C6H13 84%, R =
fluorophenyl 87%, R = -butylphenyl 83%, R = cyclohexylmethyl
81%); (c) Triethyl amine, Mesyl chloride, dry DCM, 82-97%; (d) Sodium
azide, DMF, 65 C, (87-94%); (e) CuSO4·5H2O (5 mol%), Sodium
°C, 5
p
-
synthesized
a novel series of 1-(1H-1,2,4-triazol-1-yl)-2-(2,4-
p-t
difluorophenyl)-3-(substituted or unsubstituted 1,2,3-triazol-1yl)-
propan-2-ols containing a 1,2,4-triazole ring, a difluorobenzene
group and a 1,2,3-triazole ring having long side chain or bile acid.
Some of these molecules showed much better activity than
fluconazol and amphotericin B and were found to be less toxic at
lower dose (0.001mg/mL) to human cancer cells Hep3B and
A431.14f,16
°
ascorbate (40 mol%), DMF:H2O, (4:1), Microwave (245 W), 10 min.,
(53-58%).
Scheme 1: Synthesis of compounds 6 (a-e)
the compounds in Scheme
spectroscopic method and tested for antifungal activity. The results
1 were fully characterized by
Results and Discussion
Though the 1,2,4-triazole ring, the difluorophenyl group, and the
hydroxyl group are the pharmacophores of antifungal agents,17 the
side chain also plays an important role and the optimization of the
side chain attached to the pharmacophore remains attractive to the
current research.18 Application of the isosterism concept for the
development of new compounds with therapeutic potential leads
to significant advances in molecular diversity and allows covering
chemical space in the important areas of medicinal chemistry. It is
reported that triazoles and amide group are isosteres.19 It is found
that the antifungal activity of fluconazole can be enhanced by
replacing one of the 1,2,4-triazole ring of fluconazole by isosteres
1,2,3-triazole, thiocarbamate, 1,2,4-triazolone and by incorporating
amide group or 1,2,3-triazole in the side chain.14,18
of biological activity of these compounds are summarized in Table
1.
Synthesis of fluconazole analogues in which 2, 4-disubstituted
1,2,3-triazole ring is in fluconazole component:
By using alkyne 2 we synthesized azido compound 8 using similar
reaction protocol and click reaction of compound 8 with different
alkynes 3 under microwave conditions afforded compounds 9 which
are new analogues of fluconazole containing 1,4 and 2,4-
disubstituted-1,2,3-triazole units in the side chain (Scheme 2).
Chemistry:
In continuation of our work on fluconazole analogues20 we designed
and synthesized new fluconazole analogues featuring complex side
chain having one 1,4-disubstituted 1,2,3-triazole ring and the one
2,4-disubstituted 1,2,3-triazole ring as shown in Schemes 1 and 2.
Synthesis of fluconazole analogues in which 2, 4-disubstituted
1,2,3-triazole ring is in side chain component:
Ketone 118d was reacted with propargyl bromide in the presence of
zinc to get acetylenic compound 2 as per our earlier reported16
procedure (Scheme 1). One pot reaction of alkynes 3 with
formaldehyde solution and sodium azide in glacial AcOH/ 1,4-
dioxane21 afforded alcohols 4. Mesylation of alcohols 4 followed by
reaction with sodium azide gave azides 5. Click reaction of azido
compounds 5 with alkyne 2 under microwave irradiation afforded
1,4 and 2,4-disubstituted 1,2,3-triazole containing compounds 6. All
Reagents and conditions: (a) i) AcOH, 1,4-dioxane; ii) CuSO4·5H2O (5 mol%),
Sodium ascorbate (40 mol%), 18h, 83%); (b) Triethyl amine, Mesyl chloride,
dry DCM, (82-99%); (c) Sodium azide, DMF, 65 ºC, 96%; (d) CuSO4·5H2O (5
mol%), Sodium ascorbate (40 mol%), DMF:H2O, (4:1), Microwave (245 W),
10 min., (66-75%).
Scheme 2: Synthesis of compounds 9 (a-e)
2 | J. Name., 2012, 00, 1-3
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DOI: 10.1039/C5OB00590F
Table 1. In Vitro antifungal activity of test compounds 4-9 and standard antifungal agents
Comp.No.
Minimum inhibitory concentration (MIC) µg/mL
Ca
>50
>50
50
Cn
>50
50
50
50
50
>50
50
50
50
Ss
50
>50
50
50
50
50
>50
50
50
50
1.56
0.09
0.39
50
0.19
12.5
3.12
50
6.25
25
6.25
3.12
2.0
0.12
Tm
50
>50
50
25
50
50
>50
50
25
50
3.12
0.39
1.56
0.39
6.25
50
50
50
50
50
Af
Cp
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
0.19
0.024
0.09
FP
0.003
1.56
0.19
0.78
0.19
0.39
0.09
0.39
1.0
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
12.5
1.56
12.5
1.56
12.5
>50
50
50
>50
>50
>50
>50
>50
>50
0.19
0.012
0.39
FP
0.09
6.25
0.39
0.78
0.39
1.56
0.39
0.39
0.5
50
0.012
0.00038
0.012
FP
0.00015
0.39
0.19
0.39
0.19
0.39
0.39
0.39
1.0
8
9a
9b
9c
9d
>50
>50
>50
>50
>50
2.0
50
50
1.0
0.12
9e
Fluconazole
Amphotericin B
0.12
0.06
0.5
0.12
Ca = Candida albicans; Cn = Cryptococcus neoformans; Ss = Sporothrix schenckii; Tm = Trichophyton
mentagrophytes; Af = Aspergillus fumigatus; Cp = Candida parapsilosis (ATCC-22019)
Scheme 3: Synthesis of compounds 13 (a-j)
Synthesis of amide analogues of fluconazole:
Racemic intermediate azide 11 was synthesized from ketone 1 by
our earlier reported14f procedure through epoxide 10 as shown in
Schemes 3. Reduction of Azide 11 by using hydrogen on Pd/C to
amine 12 and its coupling with different types of acids (Schemes 3)
afforded
a variety of amides 13. These amides were fully
characterized by spectroscopic analysis and were tested for their
antifungal activity. The results of biological activity are summarized
in Table 2.
Biology:
In Vitro antifungal activity of compounds 4-9 and compound
13:
All the newly synthesized compounds were tested for their
antifungal activity. The results of biological activity are summarized
in Tables 1 and 2.
Reagents and conditions: (a) Trimethylsulfoxonium iodide, NaH,
DMSO/THF, 25
H2/Pd-C, methanol, 25
DMF, 0-25 C, 10 h, (Cholic acid 92%, Isonicotinic acid 94%, Benzoic
acid 94%,22 Thiophene-2- carboxylic acid 96%,
-hydroxybenzoic acid
°
C, 2 h, 91%; (b) NaN3, DMF, 60-65 οC, 12 h, 75%; (c)
°
C, 16 h, 94%; (d) acids, EDC.HCl, HOBt, Dry
The biological activity results in Table 1 show that
°
intermediate compounds
tested fungal strains.
4
and
5
were inactive against all the
p
93%, Terphthalic acid 71%, N-boc Glycine 93%, Phenazine carboxylic
acid 96%, N- boc alanine 97%, Decanoic acid 93%).
Most of the other compounds
6
to were active against C.
9
albicans
,
C. neoformans and C. parapsilosis (ATCC-22019) than
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Table 2: In Vitro antifungal activity of compounds 13a- 13j and
standard antifungal agents
Toxicity Study:
View Article Online
DOI: 10.1039/C5OB00590F
Minimum inhibitory concentration (µg/mL)
Comp.
No.
Ca
Cn
Ss
Tm
Af
Cp
50
50
>50
50
>50
>50
>50
>50
>50
>50
>50
>50
>50
12.5
2.0
6.25
3.12
0.19
0.39
0.19
>50
>50
>50
50
13a
13b
13ca
13d
13e
13f
3.12
1.56
0.09
0.09
0.225
25
1.56
0.78
0.39
0.39
6.25
6.25
6.25
1.56
3.12
3.12
6.25
50
3.12
12.5
50
Figure 2A
Figure 2B
50
>50
>50
50
12.5
3.12
6.25
50
13g
13h
13i
50
Figure 2C
50
50
The cytotoxicity study of lead compounds 6a, 6b, 6c, 6e and 13j was
performed against mammalian fibroblast cell line (L929) by MTT
assay. Figure: 2A-Control showing normal morphology of L929 cells
at resting stage, Figure 2B- Morphology of L929 cells treated at MIC
of compound 6a (similar morphology was observed for compounds
6b, 6c, 6e and 13j at their MIC), Figure 2C-Morphology at higher
concentration (50 and 25 µg/ml) of compound 6a which was similar
to compounds 6b, 6c, 6e and 13j.
13j
0.007 0.00001
0.78
2.0
0.19
1.0
0.39
1.0
Flucona
zole
0.5
1.0
Amphot
ericinB
0.12
0.06
0.12
0.12
0.5
0.12
Ca = Candida albicans; Cn = Cryptococcus neoformans; Ss = Sporothrix
schenckii; Tm = Trichophyton mentagrophytes; Af = Aspergillus
fumigatus; Cp = Candida parapsilosis (ATCC-22019)
a Known compound22
activity particularly against yeast like fungi. These compounds were
tested for their toxicity using mouse fibroblast cell line L929.23
Morphological anomalies in L929 cells exposed to compounds 6a,
6b, 6c, 6e and 13j (25 μg/mL) were evident under phase contrast
microscope. L929 cells in control were fairly transparent and
attached to the surface of the wells of tissue culture plate (Figure
2A). The compounds did not exhibit any toxicity to L929 cells at MIC
and lower concentrations, as was evident from their normal
morphology (Figure 2B) however when exposed to higher
concentrations (50 and 25 μg/mL), the L929 cells lost their normal
morphology (Figure 2C) whereas the MTT assay revealed >90%
viability of L929 cells even at higher concentrations.
fluconazole. Some compounds were found to be more active
than amphotericin B also. Specifically compound 6b (MIC
0.00038 µg/mL for C. Neoformans) and 6e (MIC 0.00015 µg/mL
for C. Neoformans) showed very good antifungal activity and
compound 6e was found to show highest activity against
Cryptococcus neoformans (MIC 0.00015 µg/mL) and Candida
parapsilosis (ATCC-22019) (MIC 0.003 µg/mL) and hence
hemolysis and docking study was carried out on this
compound.
The biological activity results in Table 2 show that all the amides 13
(a-j) are active against C. albicans and C. neoformans. Compounds
13c and 13d showed good activity against all the strains except A.
fumigatus. Some compounds showed better activity than standard
drugs fluconazole and amphotericin B. Compound 13j showed
highest activity against all the strains, particularly against C. albicans
and C. neoformans (70 fold more active than fluconazole and 17
fold more active than amphotericin B against C. albicans while
highly active than fluconazole and amphotericin B against C.
neoformans) and hence hemolysis and docking study was carried
out on this compound.
Among all the compounds 6e and 13j were found to be most
active and may be treated as lead molecules and hence hemolysis
and docking study of these molecules was carried out.
Hemolysis Study:
Hemolytic assay results were evaluated by observing the effect of
test compounds on mammalian RBCs (Figure 3). Hemolytic assay of
compounds 6e and 13j was determined at 560 nm by using SOFT-
max Pro 4.3 Software (Molecular Devices, Sunnyvale, USA)24 and
both compounds did not cause any hemolysis at the dilutions
tested.
Among all the compounds tested for the antifungal activity,
compounds 6a, 6b, 6c, 6e and 13j were found to exhibit the best
4 | J. Name., 2012, 00, 1-3
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Conclusion:
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DOI: 10.1039/C5OB00590F
120
100
80
60
40
20
0
In conclusion we have synthesized a series of new fluconazole
analogues having complex side chain with two 1,2,3-triazole rings.
In addition we have also synthesized amide analogues of
fluconazole. All the newly synthesized molecules were found to
show good antifungal activity. Compound 6e with two 1,2,3-triazole
rings with cyclohexylmethy group in the side chain and amide
analogue of decanoic acid 13j having long chain were found to show
highest antifungal activity. These compounds did not exhibit any
toxicity to L929 cells at MIC and lower concentrations, however
when exposed to higher concentrations (50 and 25 μg/mL), the
L929 cells lost their normal morphology. The MTT assay revealed
>90% viability of L929 cells even at higher concentrations. Among
all the compounds 6e and 13j were found to be the most active and
may be treated as lead molecules and hence hemolysis and docking
study of these molecules was carried out. Both compounds 6e and
13j did not cause any hemolysis at the dilutions tested. In docking
study, overall binding mode of 6e and 13j appeared reasonable and
provide a good insight into the structural basis of inhibition of
Candida albicans Cyp51 by these compounds. This is first report of
fluconazole analogues containing 2,4-disubstituted-1,2,3-triazole
unit (Compounds 6 and 13).
6e
13j
0
20
40
60
Compound concentration (µg/ml)
Figure 3: Hemolytic assay of compounds 6e and 13j
Docking Study
Analyses of 50 possible binding poses of 6e and 13j each in the
binding site of Candida albicans Cyp51 revealed that many of
the conformations of the bound ligand are in good agreement
with the reported binding mode of flucanazole and its
analogues. Proposed binding mode of 6e and 13j in the active
site of Candida albicans Cyp51 (Figure 4) showed that in both
the molecules azole ring is optimally oriented to make
coordination bond with Fe of heme group. In docked
Experimental Protocols:
conformation of 6e
(
Figure 4A), diflurophenyl group was
Chemistry
placed near His310 and Val509. However, in case of 13j
(Figure
4B), difluorophenyl group was found to orient towards Tyr118
and Tyr132. Long hydrophobic tails of 6e and 13j occupied the
hydrophobic channel of Cyp51. We observed that binding is
mostly stabilized by hydrophobic interactions contributed by
Cyp51. This stabilization was due to the residues Phe126,
Ile131, Phe228 and Leu300. Overall binding mode of 6e and
13j appeared reasonable and provided a good insight into the
structural basis of inhibition of Candida albicans Cyp51 by
these compounds. For image generation UCSF Chimera 1.7
was used.25
All chemicals were obtained from commercial sources and used as
received without further purification. Solvents were dried according
to literature procedures. All the reactions were carried out under
nitrogen atmosphere. Column chromatography was carried out by
using silica gel (60-120 mm, Merck). All reactions were monitored
by TLC with silica gel coated plates; spots were visualized by UV
light and/or with dipping in a phosphomolybdic acid solution or
anisaldehyde solution and charring on a hot plate.1H, 13C NMR were
recorded in CDCl3 using TMS as internal standard on AC 200 MHz or
AV-400 MHz Bruker NMR spectrometers. Chemical shifts are
reported in ppm. Microwave reactions were carried in CEM
Discover, Model number: 908010. FTIR spectra were recorded on
Shimadzu FTIR-8400 spectrophotometer on KBr plate using CHCl3 or
Nujol. Only diagnostic bands are reported on cm-1 scale. The ESI ion
trap mass spectra were measured by a Finnigan MAT LCQ mass
spectrometer. The HRMS spectra were acquired on thermoscientific
Q exactive spectrometer.
General procedure for the Synthesis of 2-Substituted-1,2,3-
triazoles
CAUTION: Any experiments which may result in the formation of
hydrazoic acid should be performed in a well ventilated fume hood
and behind a blast shield. Sodium azide should not be mixed with
strong acids.
The mixture of 37% HCHO aq. (10 equiv), glacial AcOH (1.5 equiv),
and 1, 4-dioxane (5 mL) was stirred for 15 min. To this mixture was
added NaN3 (1.5 equiv), followed by alkyne (1.5 equiv). At this point
pH of the reaction mixture was 6.5. After additional 10 min of
stirring, sodium ascorbate (20 mol %) was added, followed by
Figure 4: Putative binding mode of 6e (A) and 13j (B) in the active site of
Candia albicans Cyp51. Protein is shown in mixed ribbon and stick
representation with heme prosthetic group in hot pink colour.
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CuSO4 solution (5 mol %) in 4 mL of H2O. The mixture was stirred for column chromatography on silica gel using EtOAc: Hexane (3:7) to
View Article Online
DOI: 10.1039/C5OB00590F
give pure desired products.
18h at 25 °C, diluted with H2O (30 mL) and it was extracted using
DCM (3 x 50 mL). Combined organic layer was filtered through
celite, dried over NaSO4, and concentrated on a rotary evaporator
to give crude product. The crude product was sufficiently pure to be
used for the next step without further purification.
2-(azidomethyl)-4-phenyl-2H-1,2,3-triazole (5a)
Yellow solid (88 %). IR – 2108, 1477, 1243 cm-1, 1H NMR (400 MHz,
CDCl3) δH 7.97 (s, 1H), 7.85-7.80 (m, 2H), 7.48-7.38 (m, 3H), 5.65 (s,
2H); 13C NMR (100 MHz, CDCl3) δC 149.4, 132.8, 129.5, 128.9, 126.0,
67.3
(4-Phenyl-1,2,3-triazol-1-yl)methanol (4 a)
1
Yellowish solid (92%), IR – 3276, 1640, 1456, 1296, 1077 cm-1, H
2-(azidomethyl)-4-hexyl-2H-1,2,3-triazole (5b)
1
Yellowish liquid (89 %). IR – 2929, 2108, 1519, 1242 cm-1, H NMR
NMR (400 MHz, CDCl3) δH 7.94 (s, 1H), 7.84-7.75 (m, 2H), 7.50-7.34
(m, 3H), 5.87 (d, 2H) 2H, 5.54 (t, 1H) 1H; ); 13C NMR (100 MHz,
CDCl3) δC 148.8, 147.0, 132.11, 129.6, 129.0, 128.9, 128.8, 126.1,
76.4.
(400 MHz, CDCl3) δH 7.49 (s, 1H), 5.56 (s, 2H), 2.70 (t, 2H), 1.75-1.68
(m, 2H), 1.43-1.26 (m, 6H), 0.89 (t, 3H); 13C NMR (100 MHz, CDCl3)
δC 51.0, 134.6, 66.8, 31.3, 28.8, 28.7, 25.3, 22.4, 13.9.
2-(azidomethyl)-4-(4-fluorophenyl)-2H-1,2,3-triazole (5c)
(4-hexyl-2H-1,2,3-triazol-2-yl)methanol (4b)
Yellowish liquid (84%), IR – 3310, 2929, 1519, 1081 cm- 1, 1H NMR
(400 MHz, CDCl3) δH 7.46 (s, 1H), 5.75 (s, 1H), 2.69 (t, 2H), 1.69-1.62
(m, 2H), 1.32-1.30 (m, 6H), 0.88 (t, 3H); 13C NMR (100 MHz, CDCl3)
δC 150.0, 133.7, 75.7, 31.4, 29.0, 28.8, 2.3, 22.5, 14.0.
(4-(4-fluorophenyl)-2H-1,2,3-triazol-2-yl)methanol (4c)
Yellowish solid (79%), IR – 3260, 2924, 1462, 1377, 1215, 1077cm-1,
1H NMR (400 MHz, CDCl3) δH 7.89 (s, 1H), 7.84-7.73 (m, 2H), 7.20-
7.09 (m, 2H), 5.85 (d, 2H), 4.40 (t, 1H); 13C NMR (100 MHz, CDCl3) δC
165.5, 160.6 148.0, 131.9, 127.9, 125.9, 125.8, 116.0, 76.4.
(4-(tert-butyl)phenyl)methanol (4d)
Yellowish solid, (87 %). IR – 3129, 2929, 2110, 1673, 1486, 1235,
1
978 cm-1, H NMR (400 MHz, CDCl3) δH 7.93 (s, 1H), 7.82-7.78 (m,
2H), 7.20-7.13 (m, 2H), 5.65 (s, 2H); 13C NMR (100 MHz, CDCl3) δC
132.6, 127.9, 115.0, 67.4.
2-(azidomethyl)-4-(4-(tert-butyl)phenyl)-2H-1,2,3-triazole (5d)
White solid (91 %), IR – 2964, 2107, 1618, 1486, 1242cm-1, 1H NMR
(400 MHz, CDCl3) δH 7.94 (s, 1H), 7.77 (m, 1H), 7.73 (m, 1H), 7.51
(m, 1H), 7.46 (m, 1H), 5.65 (s, 2H), 1.36 (s, 9H); 13C NMR (100 MHz,
CDCl3) δC 152.2, 149.4, 132.8, 126.7, 125.8, 67.3, 34.7, 31.2.
2-(azidomethyl)-4-(cyclohexylmethyl)-2H-1,2,3-triazole (5e)
Yellowish liquid (94 %), IR – 3444, 2925, 2105, 1622, 1242 cm-1, H
NMR (400 MHz, CDCl3) δH 7.47 (s, 1H), 5.56 (s, 1H), 3.28 (s, 1H), 2.57
(d, 2H), 1.73-1.68 (m, 5H), 1.33-0.88 (m, 6H); 13C NMR (100 MHz,
CDCl3) δC 149.4, 135.4, 66.9, 38.0, 33.1, 33.0, 26.3, 26.1.
1
White solid (83 %) IR – 3227, 2924, 1647, 1462, 1073 cm-1, 1H NMR
(400 MHz, CDCl3) δH 7.92 (s, 1H), 7.75 (m, 1H), 7.71 (m, 1H), 7.49
(m, 1H), 7.45 (m, 1H), 5.85 (d, 1H), 4.92 (t, 1H), 1.36 (s, 9H); 13C
NMR (100 MHz, CDCl3) δC 152.0, 148.7, 131.9, 126.7, 128.8, 76.2,,
34.6, 31.1.
General method for the synthesis of the compound 6 (a-e).
2-(2,4-difluorophenyl)-1-(1H-1,2,4-triazol-1-yl)pent-4-yn-2-ol
(alkyne) 2 (1 mmol) and azide 5 (a-e) (1.2 mmol) were dissolved in
DMF/H2O 4:1 (10 mL). To this solution, CuSO4.5H2O (0.05 mmol)
and sodium ascorbate (0.40 mmol) were added. The reaction
mixture was placed in a domestic microwave reactor and irradiated
for 10 min at 245 W. The reaction mixture was cooled, ice was
added, and it was then extracted with ethyl acetate. The extract
was washed with water and brine. Solvent was evaporated under
reduced pressure and the crude product was purified by column
chromatography on silica gel using 5% MeOH/CH2Cl2 system to
obtain 1,4-disubstituted 1,2,3-triazole compounds 6 (a-e).
(4-(cyclohexylmethyl)-2H-1,2,3-triazol-2-yl)methanol (4e)
Colorless liquid, IR – 3111, 2924, 1516, 1449, 1080 cm-1, 1H NMR
(400 MHz, CDCl3) δH 7.47 (s, 1H), 5.56 (s, 2H), 3.28 (s, 1H), 2.58 (d,
2H), 1.72-1.65 (m, 5H), 1.33-0.88 (m, 6H); 13C NMR (100 MHz, CDCl3)
δC 148.6, 134.4, 75.8, 38.0, 33.1, 32.9, 32.7, 26.2, 26.1.
General procedure for the Synthesis of 2-azidomethyl-1,2,3-
triazoles
The solution of 2-substituted-1,2,3-triazoles obtained in first step in
dry DCM (5 mL) was cooled to 0 °C. Triethyl amine (1.5 equiv) was
added and the reaction mixture was stirred for 5 min at 0 °C. To this
mixture methane sulfonyl chloride (3 equiv) was added and the
reaction mixture was stirred at 0 °C for 2h. It was then diluted with
cold H2O and extracted with DCM. Organic layer was washed with
dilute NaHCO3, brine and dried over Sodium sulfate. It was then
filtered and concentrated under vacuum to give crude product
which was used for next step without further purification.
2-(2,4-difluorophenyl)-1-(1-((4-phenyl-2H-1,2,3-triazol-2-yl)methyl)-
1H-1,2,3-triazol-4-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (6a)
Yellowish solid (58 %), IR – 3421, 1617, 1500, 1456, 1138, 965 cm-1,
1H NMR (400 MHz, CDCl3) δH 8.10 (s, 1H), 7.90 (s, 1H), 7.80 (s, 1H),
Compound obtained in the above step was dissolve in dry DMF. 7.76 (d, J = 7.34 Hz, 2H), 7.52 (s, 1H), 7.45 (t, J= 7.02, 2H), 7.40 (t,
Sodium azide (3 equiv) was added and the reaction mixture was 1H), 7.31-7.26 (m, 1H), 6.72 (dd, J = 14.03 & 19.53 Hz, 2H), 6.69-
6.64 (m, 1H), 6.61-6.57 (m, 1H), 5.25 (bs, 1H), 4.71 (d, J = 14.34, 1H),
4.55 (d, J = 14.34 Hz, 1H), 3.45 (d, J = 14.95 Hz, 1H), 3.15 (d, J =
15.26 Hz, 1H); 13C NMR (100 MHz, CDCl3) δC 163.5, 161.5, 159.4,
157.38, 145.0, 143.6, 133.3, 129.9, 129.3, 129.0, 126.1, 124.4,
122.7, 111.5, 103.9, 75.2, 64.8, 56.8, 33.9; HRMS calcd for
C22H20N9OF2 [M + H]+: 464.1753, found: 464.1749.
stirred at 65 °C for 6h. After cooling to room temperature, the
reaction mixture was diluted with cold water and was extracted
with DCM (3X50 mL). Combined organic layer was washed with
water and brine, dried over sodium sulfate and concentrated on
rotary evaporator. The crude mixture obtained was purified by
6 | J. Name., 2012, 00, 1-3
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Organic & Biomolecular Chemistry
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Journal Name
COMMUNICATION
2-(2,4-difluorophenyl)-1-(1-((4-hexyl-2H-1,2,3-triazol-2-yl)methyl)-
1H-1,2,3-triazol-4-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (6b)
(m, 2H), 6.83-6.68 (m, 2H), 5.67 (s, 2H), 4.97 (bs, 1H), 4.77 (d, J =
View Article Online
DOI: 10.1039/C5OB00590F
14.15 Hz, 1H), 4.56 (d, J = 14.15 Hz), 3.44 (d, J = 14.15 Hz, 1H) 3.21
Glassy solid (55%); IR -3418, 1617, 1502, 1458, 1145, 965 cm-1, 1H (d, J = 14.15 Hz, 1H); HRMS calcd for C14H15N6O2F2 [M + H]+:
NMR (400 MHz, CDCl3) δH 8.02 (s, 1H), 7.73 (s, 1H), 7.40-7.35 (m, 337.1219, found: 337.1046.
2H), 7.24-7.29 (m, 2H), 6.66-6.54 (m, 2H), 5.18 (s, 1H), 4.64 (d, J =
14.34 Hz, 1H), 4.45 (d, J = 14.34 Hz, 1H), 3.37 (d, J= 13.84 Hz, 1H), 1-(2-(azidomethyl)-2H-1,2,3-triazol-4-yl)-2-(2,4-difluorophenyl)-3-
3.05 (d, J = 13.84 Hz, 1H), 2.56 (t, 2H), 1.55 (m, 2H), 1.25-1.19 (m, (1H-1,2,4-triazol-1-yl)propan-2-ol (8)
6H), 0.81 (t, 3H); 13C NMR (100 MHz, CDCl3 + MEOD4) δC 164.7, Yellowish solid (96 %), IR – 3132, 3016, 2112, 1618, 1501, 1216 cm -
160.8, 159.7, 155.7, 150.9, 150.3, 142.9, 134.8, 129.4, 123.9, 122.8, 1, 1H NMR (400 MHz, CDCl3) δH 8.02 (s, 1H), 7.83 (s, 1H), 7.40 (s, 1H),
111.0, 103.6, 74.3, 64.05, 56.9, 33.8, 31.0, 29.3, 28.4, 24.9, 22.1, 7.37-7.32 (m, 1H), 7.80-7.70 (m, 2H), 5.49 (s, 2H), 4.94 (bs, 1H), 4.80
13.5; HRMS calcd for C22H28N9OF2 [M + H]+: 472.2379, found: (d, J = 14.20 Hz, 1H), 4.53 (d, J = 14.20 Hz, 1H), 3.46 (d, J = 15.57 Hz,
472.2377.
1H), 3.19 (d, J = 14.20 Hz, 1H); 13C NMR (100 MHz, CDCl3) δC 151.5,
144.8, 144.2, 136.3, 129.7, 124.1, 111.5, 104.0, 75.0, 68.8, 56.6,
34.3; HRMS calcd for C14H14N9OF2 [M + H]+: 362.1284, found:
362.1280.
2-(2,4-difluorophenyl)-1-(1-((4-(4-fluorophenyl)-2H-1,2,3-triazol-2-
yl)methyl)-1H-1,2,3-triazol-4-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol
(6c)
1
Glassy solid (59%); H NMR (400 MHz, CDCl3) δH 8.09 (s, 1H), 7.86
(s,1H), 7.79-7.70 (m, 3H), 7.52 (s, 1H), 7.36-7.28 (m, 1H), 7.19-7.10 General method for the synthesis of the compound 9 (a-e):
(m, 2H), 6.72 (d, 2H), 6.68-6.55 (m, 2H), 5.23 (s, H), 4.73 (d, J = 14.15 By using alkyne 8 (1 mmol) and azide 3 (a-e) (1.2 mmol) and
Hz, 1H), 4.54 (d, J = 14.15 Hz, 1H), 3.46 (d, J = 15.79 Hz, 1H), 3.15 (d, following procedure for click reaction as for compounds 6,
J = 15.79 Hz, 1H); 13C NMR (100 MHz, CDCl3) δC : 164.3, 163.5, compound 9 (a-e) were obtained.
162.3, 161.6, 19.4, 157.4, 151.6, 149.1, 143.7, 133.1, 129.1, 128.0,
125.4, 124.4, 122.7, 116.1, 111.4, 104.0, 75.2, 64.8, 56.8, 33.9; 2-(2,4-difluorophenyl)-1-(2-((4-phenyl-1H-1,2,3-triazol-1-yl)methyl)-
HRMS calcd for C22H19N9OF3 [M + H]+: 482.1659, found: 482.1655.
1-(1-((4-(4-(tert-butyl)phenyl)-2H-1,2,3-triazol-2-yl)methyl)-1H-
1,2,3-triazol-4-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-
yl)propan-2-ol (6d)
2H-1,2,3-triazol-4-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (9a)
Yellowish solid (68 %), H NMR (400 MHz, CDCl3) δH 8.02 (s, 1H),
7.85 (s, 1H), 7.83 (s, 1H), 7.79 (d, J = 7.27 Hz, 2H), 7.44-7.41 (m, 3H),
7.37-7.33 (m, 1H), 7.25-7.20 (m, 1H), 6.76-6.69 (m, 3H), 6.61-6.56
1
Gummy solid (57 %), IR – 3400, 2964, 1617, 1499, 1272, 1137 cm-1, (m, 1H), 4.99 (bs, 1H), 4.77 (d, J = 14.03 Hz, 1H), 4.52 (d, J = 14.03
1H NMR (400 MHz, CDCl3) δH 8.08 (s, 1H), 7.88 (s, 1H), 7.80 (s, 1H), Hz, 1H), 3.43 (d, J = 14.80 Hz, 1H), 3.17 (d, J = 14.80 Hz, 1H); 13C
7.72(m, 1H), 7.67 (m, 1H), 7.50 (dd, J = 7.4Hz, 2H), 7.45 (s, 1H), 7.36- NMR (100 MHz, CDCl3) δC 163.9, 161.4, 159.5, 157.0, 152.0, 148.8,
7.28 (m, 1H), 7.71 (d, J = 6.86 Hz, 2H), 6.68-6.55 (m, 2H), 5.23 (s, 145.5, 137.0, 129.8, 128.9, 128.6, 125.8, 124.0, 119.2, 111.5, 104.1,
1H), 4.63 (d, J = 4.43Hz, 1H), 4.54 (d, 4.5 Hz, 1H), 3.45 (d, J = 3.5Hz, 75.0, 64.2, 56.6, 34.4; HRMS calcd for C22H20N9OF2 [M + H]+:
1H), 3.14 (d, J = 3.18Hz, 1H), 1.35 (s, 9H); 13C NMR (100 MHz, CDCl3) 464.1753, found: 464.1749.
δC : 164.9, 160.7, 159.9, 155.8, 152.4, 149.8,143.4, 133.1, 129.8, 2-(2,4-difluorophenyl)-1-(2-((4-hexyl-1H-1,2,3-triazol-1-yl)methyl)-
126.1, 125.8, 124.4, 122.7, 111.1, 103.8, 77.2, 75.0, 64.7, 71.7, 34.6, 2H-1,2,3-triazol-4-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (9b)
1
33.9, 31.1; HRMS calcd for C26H28N9OF2 [M + H]+: 520.2379, found: Yellowish solid (75 %), H NMR (CDCL3) δH
: 8.01 (s, 1H), 7.83 (s,
520.2379.
1H), 7.37 (dd, J = 7.96 Hz, 2H), 6.80-6.63 (m, 4H), 4.94 (s, 1H), 4.77
(d, J = 14.51 Hz, 1H), 4.49 (d, J = 14.51 Hz, 1H), 3.42 (d, J = 14.78 Hz,
1-(1-((4-(cyclohexylmethyl)-2H-1,2,3-triazol-2-yl)methyl)-1H-1,2,3-
triazol-4-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2- 1H), 3.15 (d, J =14.78 Hz, 1H), 2.67 (t, 2H), 1.62-1.58 (m, 2H), 1.29-
ol (6e)
1.25 (m, 6H), 0.87 (t, 3H); 13C NMR (100 MHz, CDCl3) δC : 163.8,
Glassy solid (58 %) IR – 3422, 3019, 2927, 1618, 1500, 1215 cm-1, 1H 161.3, 159.5, 157.1, 149.4, 145.3, 136.7, 132.8, 124.1, 111.5,
NMR (400 MHz, CDCl3) δH 8.09 (s, 1H), 7.80 (s, 1H), 7.46 (s, 1H), 104.0,77.3, 74.9, 64.4, 60.4, 56.6, 34.3, 31.4, 29.1, 28.8, 25.4, 22.4,
7.41 (s, 1H), 7.38-7.29 (m, 1H), 6.76-6.65 (m, 2H), 6.62 (d, J = 2.15 22.3, 14.0; HRMS calcd for C22H28N9OF2 [M + H]+: 472.2379, found:
Hz, 2H), 5.25 (s, 1H), 4.72 (d, J = 14.27 Hz, 1H), 4.53 (d, J = 14.27 Hz, 472.2377.
1H), 3.44 (d, J = 13.89 Hz, 1H), 3.14 (d, J = 13.89 Hz, 1H), 2.53 (d, 2-(2,4-difluorophenyl)-1-(2-((4-(4-fluorophenyl)-1H-1,2,3-triazol-1-
2H), 1.77-1.71 (m, 3H), 1.70-1.64 (m, 3H), 1.21-1.13 (m, 2H), 1.05- yl)methyl)-2H-1,2,3-triazol-4-yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol
0.85 (m, 2H); 13C NMR (100 MHz, CDCl3) δC 164.8 160.7, 159.8, (9c)
155.7, 149.7, 143.2, 135.2, 129.8, 124.4,122.5, 111.05, 103.7, Sticky solid (71 %), IR- 3419, 2925, 1616, 1499, 1231, 966 cm-1, 1H
77.2,74.8, 64.3, 56.8, 37.6, 33.8, 32.8, 32.7, 29.4, 26.0, 25.8; HRMS NMR (400 MHz, CDCl3) δH 8.01 (s, 1H), 7.84-7.70 (m, 4H), 7.43 (s,
calcd for C23H28N9OF2 [M + H]+: 484.2379, found: 484.2380.
1H), 7.22-7.08 (m, 2H), 6.80-6.54 (m, 4H), 4.96 (bs, 1H), 4.78 (d, J =
14.02 Hz, 1H), 4.51 (d, J = 14.02 Hz, 1H), 3.44 (d, J = 14.78 Hz, 1H),
3.16 (d, J = 14.78 Hz, 1H); HRMS calcd for C22H19N9OF2 [M + H]+:
482.1659, found: 464.1655.
2-(2,4-difluorophenyl)-1-(3-(hydroxymethyl)cyclopenta-1,4-dien-1-
yl)-3-(1H-1,2,4-triazol-1-yl)propan-2-ol (7)
Yellowish solid (83 %) IR – 3420, 3020, 2927, 1617, 1499, 1216 cm-
1,1H NMR (400 MHz, CDCl3) δH 8.06 (s, 1H), 7.82 (s, 1H), 7.40-7.31
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Organic & Biomolecular Chemistry
Page 8 of 11
COMMUNICATION
Journal Name
1-(2-((1-(4-(tert-butyl)phenyl)-1H-1,2,3-triazol-4-yl)methyl)-2H-
1,2,3-triazol-4-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-
yl)propan-2-ol (9d)
View Article Online
DOI: 10.1039/C5OB00590F
(4R)-N-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-
yl)propyl)-4-((3R,5S,7R,8R,
9S,10S,
12S,13R,14S,17R)-3,7,12-
Yellowish solid (69 %), IR – 3130, 2963, 1667, 1617, 1500, 1272, trihydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta
1138 cm-1, 1H NMR (400 MHz, CDCl3) δH 8.09 (s, 1H), 7.87 (s, 1H), [a]phenanthren-17-yl)pentanamide(13a)
7.78 (s, 1H), 7.68 (d, J = 8.24 Hz, 2H), 7.50 (s, 1H), 7.47 (d, J = 8.24 White solid, (92 %); 1H NMR (400 MHz, CDCL3) δH - 8.20 (d, 1H), 7.79
Hz, 2H), 7.32-7.28 (m, 1H), 6.71 (dd, J = 13.73 & 20.45 Hz, 2H), 6.68- (s, 1H), 7.62-7.54 (m, 1H), 7.39 (d, 1H), 6.83-6.75 (m, 2H), 4.60-4.51
6.57 (m, 2H), 5.26 (s, 1H), 4.72 (d, J = 14.04 Hz, 1H), 4.55 (d, J = (m, 2H), 3.88 (s, 1H), 3.81(s, 1H), 3.68 (bs, 2H), 3.37(s, 1H), 3.06 (m,
14.04 Hz, 1H), 3.45 (d, J = 14.91 Hz, 1H), 3.15 (d, J = 14.95 Hz, 1H), 1H), 0.87 (d, 6H), 0.62 (s, 3H); 13C NMR (100 MHz, CDCL3) δC 177.5,
1.35 (s, 9H); 13C NMR (100 MHz, CDCl3) δC 163.5, 161.5, 159.3, 161.5, 150.9, 144.7, 130.4, 124.0, 118.5, 111.4, 104.0, 76.1, 73.0,
157.4, 152.5, 151.4, 149.9, 143.6, 133.2, 129.9, 126.2, 125.9, 124.4, 71.7, 56.2, 47.4, 46.2, 45.6, 41.5, 39.3, 35.5, 34.7, 31.9, 31.4, 29.6,
122.6, 111.4, 103.9, 75.1, 64.8, 56.8, 34.7, 33.9, 31.2; HRMS calcd 29.3, 26.4, 22.6, 22.4, 17.2, 14.1, 12.4, 8.8. HRMS calcd for
for C26H28N9OF2 [M + H]+: 520.2379, found: 520.2379.
C35H51N4O5F2 [M + H]+: 645.3823, found: 645.3823.
1-(2-((4-(cyclohexylmethyl)-1H-1,2,3-triazol-1-yl)methyl)-2H-1,2,3-
N-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)
triazol-4-yl)-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2- isonicotinamide (13b)
ol (9e)
Brown sticky solid (94 %); 1H NMR (400 MHz, DMSO d6) δH 8.69 (t,
Yellowish solid (72 %), IR – 3661, 2926, 1667, 1617, 1500, 1273, 1H), 8.63 (s, 1H), 8.28 (s, 1H), 7.70(s, 1H), 7.60-7.56 (m, 1H), 7.37-
1
1138 cm-1, H NMR (400 MHz, CDCl3) δH 8.03 (s) 1H, 7.83 (s) 1H, 7.31 (m, 1H), 7.11-7.06 (m, 1H), 6.88-6.84 (m, 1H), 6.15 (s, 1H), 4.68
7.38 (s, 1H) 7.33 (s, 1H) , 7.29-7.23 (m) 1H, 6.77-6.66 (m) 2H, 6.63 (d, J = 14.43 Hz, 1H), 4.55 (d, J = 14.43 Hz, 1H), 3.82-3.77 (dd, 2H);
(S, 2H), 4.97 (s, 1H), 4.77 (d, J = 13.94 Hz), 4.51 (d, J = 13.94 Hz) 1H, 13C NMR (100 MHz, DMSO d6) δC 166.5, 163.5, 161.1, 158.4, 150.9,
3.42 (d, J = 14.67 Hz), 3.16 (d, J = 14.67 Hz), 2.54 (d, J = 6.78 Hz) 2H, 150.5, 145.5, 141.6, 130.4, 124.9, 121.8, 111.2, 104.4, 79.5, 75.4,
1.67-1.57 (m) 5H, 1.20-1.08 (m) 3H, 0.96-0.82 (m) 3H; 13C NMR (100 55.5, 46.9; HRMS calcd for C17H16N5O2F2 [M + H]+: 360.1266, found:
MHz, CDCl3) δC 164.7, 160.7, 159.7, 155.7, 149.6, 143.2, 135.6, 360.1267.
129.8, 124.3, 122.5, 111.3, 110.8, 103.7, 74.8, 64.2, 56.8, 37.5, 33.8, N-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-
32.7, 32.7, 26.0, 25.8; HRMS calcd for C22H20N9OF2 [M + H]+: yl)propyl)benzamide (13c)22
1
484.2379, found: 484.2380.
Yellowish solid, (94 %); H NMR (400 MHz, DMSO d6) δH 8.53 (s,
1H), 8.35 (s, 1H), 7.75-7.72 (m, 3H), 7.51-7.48 (m, 1H), 7.43-7.40 (m,
3H), 7.16-7.10 (m, 1H), 6.92-6.88 (m, 1H), 6.36 (bs, 1H), 4.68 (d, J =
14.43 Hz, 1H), 4.59 (d, J = 14.43 Hz, 1H), 3.86 (d, J = 6.11 Hz, 1H),
1-amino-2-(2,4-difluorophenyl)-3-(1H-1,2,4-triazol-1-yl)propan-2-
ol (12)
To a solution of 1-azido-2-(2,4-difluorophenyl)propan-2-ol (3.5 g, 3.76 (d, J = 6.11 Hz, 1H); 13C NMR (100 MHz, DMSO d6) δC 168.4,
12.5 mmol) in 40 mL of ethanol was added 10% active charcoal- 163.5, 161.0, 158.3, 150.9, 145.5, 134.2, 132.0, 130.5, 129.8, 128.8,
supported palladium (350 mg). The solution was stirred overnight at 127.7, 125.2, 115.3, 111.2, 104.4, 75.6, 55.7, 47.1; HRMS calcd for
room temperature under a hydrogen atmosphere (60 psi) and C18H17N4O2F2 [M + H]+: 359.1313, found: 359.1314.
filtered through celite. It was then concentrated, the residue was N-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-
washed with ethyl acetate-pet ether (50:50) and oven dried to get yl)propyl)thiophene-2-carboxamide (13d)
the desired product 12.
White solid, (96 %); 1H NMR (400 MHz, Methanol D4) δH - 8.7 (s, 1H),
7.78 (s, 1H), 7.64-7.60 (m, 1H), 7.53-7.47 (m, 1H), 7.08 (t, 1H), 6.97-
6.91 (m, 1H), 6.85-6.81 (m, 1H), 5.49 (s, 1H), 4.79 (d, J = 14 Hz, 1H),
4.67 (d, J = 14 Hz, 1H), 3.89 (dd, 2H); 13C NMR (100 MHz,
METHANOL-D4) δC 166.1, 165.7, 163.2, 159.7, 151.4, 146.3, 139.2,
132.4, 131.5, 130.3, 129.0, 125.6, 112.1, 105.1, 77.1, 57.2, 54.9,
48.4; HRMS calcd for C17H12N4O2F2Na[M + H]+: 365.0881, found:
365.0821.
Yellowish solid (94 %), mp. 253 °C; IR (Nujol): 3315.98, 3115.38,
2922.61, 2851.64, 1616.12, 1598.42,1455.82,1272.03 cm-1. H NMR
1
(400 MHz, CDCL3) δH 8.07 (s, 1H), 7.81 (s, 1H), 7.48-7.41 (m, 1H),
6.90-6.73 (m, 2H), 4.85 (s, 2H), 3.20 (d, J = 13.19 Hz, 1H), 2.97 (d, J =
13.19 Hz, 1H); 13C NMR (100 MHz, CDCL3) δC 164.1, 161.6, 150.9,
144.6, 130.0, 124.6, 111.5, 104.2, 74.7, 57.3, 33.8; LCMS for
C11H13N4OF2 [M + H]+: 287.4.
N-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-yl)propyl)-
4-hydroxybenzamide (13e)
General procedure for the synthesis fluconazole containing amide:
An acid (1 mmol) and Fluconazole amine 12 (1 mmol) were
dissolved in dry DMF under argon atmosphere and the solution was
cooled to 0 °C. HOBt (0.5 mmol) and EDC.HCl (0.5 mmol) were
added and stirring was continued for 30 minute. The reaction
mixture was allowed to attain room temperature and it was stirred
further for 10 h. The reaction was quenched by adding ice and
extracted with ethyl acetate (3 times). Organic layer was washed
with water and brine dried over sodium sulfate and evaporated to
get the desired product.
Yellowish solid, (93 %); 1H NMR (400 MHz, DMSO d6) δH 8.15(s,
1H), 7.96 (s, 1H), 7.76 (s, 1H), 7.63-7.54 (m, 2H), 7.49 (dd, J = 1.14
Hz, 1H), 7.06-7.01 (m, 1H), 6.86-6.74 (m, 2H), 6.29 (s, 1H), 5.33 (s,
1H), 4.64 (dd, J = 14.34 Hz, 2H), 3.97-3.76 (m, 2H); 13C NMR (100
MHz, CDCl3+MeOD-D4) δC - 170.3, 160.5,150.0, 144.3, 132.2, 129.8,
128.8, 128.3, 123.5, 122.0, 115.1, 114.8, 111.9, 103.6, 75.6, 55.7,
47.1; HRMS calcd for C18H17N4O3F2 [M + H]+: 375.1263, found:
375.1265.
8 | J. Name., 2012, 00, 1-3
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N1,N4-bis(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-
Trichophyton mentagrophytes, Aspergillus fumigatVuise,w ArtCicalenOdnildinae
DOI: 10.1039/C5OB00590F
yl)propyl)terephthala mide (13f)
parapsilosis (ATCC-22019) using fluconazole and amphotericin B as
1
Yellowish solid, (71 %); H NMR (400 MHz, CDCL3) δH 8.41 (s, 2H), standard drugs. Minimum inhibitory concentration (MIC) values
7.81 (s, 2H), 7.53-7.48 (m, 2H), 6.97-6.93 (m, 2H), 6.86-6.82 (m, 2H), were determined using standard broth microdilution technique as
4.81 (d, J = 14.34 Hz, 2H), 4.78 (d, J = 14.34 Hz, 2H), 3.98 (d, J = per NCCLS guidelines25. The results of biological activity are
14.34 Hz, 2H), 3.88 (d, J = 14.34 Hz, 2H); 13C NMR (100 MHz, CDCL3) summarized in Tables 1 and 2.
δC 167.8, 162.3, 160.4, 158.9, 156.8, 148.5, 135.0, 128.4, 125.6, Materials and Methods: MIC determination: Minimum inhibitory
122.4, 117.1, 109.1, 108.1, 102.1, 74.0, 54.1, 27.8; HRMS calcd for concentration (MIC) of compounds was tested according to
C30H27N8O4F4 [M + H]+: 639.2086, found: 609.2089.
standard microbroth dilution technique as per NCCLS guidelines.
tert-butyl [2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1- Briefly, testing was performed in flat bottom 96 well tissue culture
yl)propyl)amino)-2-oxoethyl]carbamate (13g)
plates (CELLSTAR_ Greiner bio-one GmbH, Germany) in RPMI 1640
Yellowish solid, (93 %); 1H NMR (400 MHz, METHANOL-D4) δH - 8.36 medium buffered with MOPS (3-[N- morpholino]propanesulfonic
(s, 1H), 7.79 (s, 1H), 7.49 (m, 1H), 6.98-6.94 (m, 1H), 6.88-6.86 (m, acid) (Sigma Chem. Co., MO, USA) for fungal strains.
1H), 4.77-4.75 (d, J = 14.34 Hz, 1H), 4.65-4.62 (d, J = 14.34 Hz, 1H),
3.88-3.86 (d, J = 14.34 Hz, 1H), 3.74-3.71 (d, J = 14.34 Hz, 1H), 3.68-
3.64 (m, 2H), 1.46 (s, 9H); 13C NMR (100 MHz, METHANOL-D4) δC
172.5, 164.0, 162.0, 160.4, 158.41, 157.0, 150.0, 144.8, 130.00,
123.9, 110.7, 103.6, 79.5, 55.4, 46.0, 43.2, 27.3; HRMS calcd for
C18H24N5OF2 [M + H]+: 412.1791, found: 412.1790.
The concentration range of test compounds was 50–0.36 μg/mL
and for standard compounds 32–0.0018 μg/mL. The plates were
incubated in a moist chamber at 35 °C and observed absorbance
were after 24 h for C. albicans, 48h for C. parapsilosis and
Cryptococcus neoformans, 72
h for Aspergillus fumigatus, S.
schenckii, and Trichophyton mentagrophytes. MIC was determined
N-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-
yl)propyl)phenazine-1-carboxamide (13h)
as 80% inhibition of growth with respect to the growth control.
Yellowish solid, (96 %); 1H NMR (400 MHz, CDCL3) δH – 10.57(t, 1H),
8.30 (dd, J = 8Hz & 1.2 Hz, 1H), 7.82 (dd, J = 8Hz & 1.2 Hz, 1H), 7.71
(dd, J = 8Hz & 1.2 Hz, 1H), 7.61 (s, 1H), 7.57 (dd, J = 8Hz & 1.2 Hz,
1H), 7.43-7.33 (m, 3H), 7.16-7.10 (m, 1H), 6.71 (s, 1H), 6.25-6.16 (m,
2H), 5.83 (s, 1H), 4.14 (dd, 2H), 3.62 (dd, 2H); 13C NMR (100 MHz,
CDCL3) δC 167.5, 164.0, 161.5, 160.0, 157.6, 151.4, 144.6, 143.3,
141.1, 140.4, 135.5, 138.4, 132.1, 131.2, 130.6, 129.8, 128.8,,127.6,
124.3, 111.6, 103.9, 76.9, 56.5, 47.9; HRMS calcd for C24H19N6O2F2
[M + H]+: 461.1532, found: 461.1534.
Toxicity Study:
Compounds 6a, 6b, 6c, 6e and 13j were found to exhibit the best in
vitro activity against all the fungi. These compounds were tested for
their toxicity using mouse fibroblast cell line L929.
Material and Method
To test the toxicity of lead compounds 6a, 6b, 6c, 6e and 13j
against mammalian cells, mouse fibroblast cell line L929 was
used. Stock solutions (1mg/mL) of the test compounds were
prepared in DMSO. The cell line L929 was grown in RPMI 1640
medium supplemented with 10% FBS and 1 X antimycotic
solution (sigma, USA) at 37 oC in humidified atmosphere
having 5% CO2. One hundred μL (1x103 cells µL in RPMI) of the
confluent fibroblast stock suspension (1x105 cells/mL) was
dispensed in 96 well tissue culture plates. The original medium
from the wells was replaced with 100 µL serum free RPMI
when the cells reached 80% confluence after incubation in a
CO2 incubator at 37 oC. Various concentrations of the test
compounds (25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.19, 0.09
μg/mL) were added to the growing cells along with control
(with no compounds) and incubated for 24 hours. 200 µL of
MTT solution (0.5 mg of MTT in RPMI 1640 medium) was
added to each well after removing the media completely and
tert-butyl (1-((2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-
1-yl)propyl)amino)-1-oxopropan-2-yl)carbamate (13i)
1
Brown sticky solid, (97 %); H NMR (400 MHz, CDCL3) δH 8.07 (d,
1H), 7.83 (s, 1H), 7.56-7.53 (m, 1H), 6.82-6.76 (m, 2H), 5.75(d, 1H),
4.83(bs, 1H), 4.66-4.1 (m, 2H), 4.06-4.02(m, 1H), 3.88-3.78(m, 1H),
3.67-3.57 (m, 1H), 1.42 (d, 9H), 1.22-1.19 (m, 3H); 13C NMR (100
MHz, CDCL3) δC 175.1, 151.7, 144.6, 130.2, 123.7, 111.7, 104.1,
76.3, 55.5, 50.0, 46.9, 29.7, 28.2, 17.9; HRMS calcd for C19H26N5O4F2
[M + H]+: 426.1947, found: 426.1942.
N-(2-(2,4-difluorophenyl)-2-hydroxy-3-(1H-1,2,4-triazol-1-
yl)propyl)decanamide (13j)
Brown sticky solid, (93 %); 1H NMR (400 MHz, CDCL3) δH 8.23 (s, 1H),
7.86 (s, 1H), 7.62-7.57 (m, 1H), 6.86-6.78 (m, 2H), 6.0 (bs, 1H), 4.55
(s, 1H), 3.76-3.67 (m, 2H), 2.12-2.00 (m, 2H), 1.47-1.41 (m, 2H),
1.29-1.19 (m, 10H), 1.13-1.11 (m, 2H), 0.88 (t, 3H); 13C NMR (100
MHz, CDCL3) δC 176.2, 163.9, 162.00, 159.7, 157.7, 150.9, 130.4,
123.7, 111.8, 104.0, 76.2, 56.2, 47.5, 36.1, 31.8 29.3, 29.2, 28.9,
25.5, 22.6, 14.0; LCMS for C17H16N5O2F2 [M + H]+: 385.1.
o
incubated for 4 hours at 37 C to allow MTT metabolism. An
aliquot of 100 µL of DMSO solvent was added to each well and
the plate was incubated for 30 minutes at room temperature.
Response of L929 cells to the test compounds was determined
spectrophotometrically at 570 and 630 nm. The morphology of
the cells was observed using Giemsa stain under Phase
contrast microscope. After fixation of the cells in the wells of
96 well tissue culture plates, Giemsa stain was added to each
Biology:
In Vitro antifungal activity
All the newly synthesized compounds were tested for their in vitro
antifungal activity against different fungal strains such as Candida
albicans, Cryptococcus neoformans, Sporothrix schenckii,
o
well and incubated for 30 min at 37 C. The excess stain was
removed by thorough washing with phosphate buffer saline
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and the culture plates were air dried and observed under a
phase contrast microscope.
View Article Online
DOI: 10.1039/C5OB00590F
Notes and references
Hemolysis Study:
Hemolytic assay was done by observing the effect of test
compounds on mammalian RBCs.
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collected by centrifugation at 1000g for 5 min. Supernatant was
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Acknowledgement:
Authors thank Indian Council for Medical Research for financial
support (Project no. 58/24/2007-BMS) and CSIR, New Delhi for
financial support under ORIGIN (CSC-0108) and OSDD (HCP-0001).
SGA ((31/11(802)/2013-EMR-I) thank CSIR, New Delhi for a senior
research fellowship.
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